Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1

Abstract

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed alpha and beta subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the alpha subunit formed a dimeric structure and behaved as an NADH dehydrogenase; beta subunit was a tetramer that had sarcosine oxidase and l-proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (alpha beta)(4) form and had NADH dehydrogenase activity. Gold-label analysis indicated that alpha and beta subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (alpha beta)(4) complex may separately exist as a function enzyme in different conditions.